Neutralization of Macrophage Migration Inhibitory Factor (MIF) can Prevent Proliferative Vitreoretinopathy (PVR) by Inhibiting Epithelio-Mesenchymal Transformation (EMT) of Retinal Pigment Epithelium (RPE)

Abstract

Purpose: : To determine whether MIF inhibition can prevent the formation PVR by blocking EMT of RPE.

Methods: : Primary human RPE cells from two young donors (20 and 26 years old) were plated on tissue-culture plastic and maintained in DMEM H16 supplemented with 10%FBS and a MIF inhibitor (4-IPP, 25-50 µM) for 3 weeks. Control cells were exposed only to the solvent (DMSO/Corn oil). Morphology and qPCR for microphthalmia transcription factor (MITF), plasminogen activator inhibitor type-1 (PAI-1), vimentin and N-cadherin was used to asses the presence of EMT. The effect of MIF inhibition on EMT was further tested in vivo using an animal model of PVR. 6 adult C57BL/6 mice were anesthetized with an IP pentobarbital sodium (70 mg/kg) and a linear incision was made in the cornea. The lens was removed and a tear was created in the peripheral retina using a 32G needle. Corneal incision was then sutured with 10-0 nylon. Experimental group received 500 µg/in 5 µl of a MIF inhibitor (ACT-003) in the posterior subtenon space on Days 0 and 5 whereas control animals received the solvent. Animals were sacrificed on Day 10 and light microscopy was used to asses the presence and extend of PVR. MIF -/- animals (n=3) were also used as control.

Results: : Cultured human RPE cells treated with MIF-inhibitor were significantly fewer in number but maintained their typical cuboidal epithelial morphology, in contrast to control RPE that acquired a fibroblast-like morphology. Consistent with the cell morphology, MIF neutralization resulted in the reduction of mRNA levels of mesenchymal markers (vimentin, 8.5x; PAI-1, 19.7x and N-cadherin, 43,2x) and an increase in mRNA levels of the epithelial gene product, MITF (1.6x). In control eyes, surgically-induced retinal detachment was complicated with the formation of epi- and subretinal fibrotic membranes, as well as multilayering of the RPE cells. RPE cells remained as a monolayer in the experimental group that received MIF-inhibitor and in MIF -/- animals. Neutralization of MIF significantly inhibited the formation of peri-retinal membranes.

Conclusions: : MIF is an important regulator of EMT in RPE. Neutralization of MIF is a potential therapeutic target for the prevention and treatment of ocular proliferative disorders such as PVR.

Keywords: proliferative vitreoretinopathy • retinal pigment epithelium • wound healing